The invention relates to an optical device with a focusing optic which focuses a minimum of one light beam in a focal plane, and a phase filter to form the focus of the light beam such that the phase filter effects a phase shift of the light beam.
The invention further relates to a scanning microscope with a focusing optic which focuses a light beam in a focal plane, and with a phase filter to form the focus of the light beam such that the phase filter effects a phase shift of the light beam.
In scanning microscopy, a sample is illuminated with a light beam for the purpose of observing the reflected or fluorescent light emitted by the sample. The focus of the illumination light beam is moved in an object plane with the help of a controllable beam deflection device (scanning device), generally by tilting two minors, with the deflection axes usually positioned perpendicular to each other so that one minor deflects in the x-direction and the other in the y-direction.
A confocal scanning microscope generally comprises a light source, a focusing optic with which the light from the source is focused on a pinhole aperture, a beam splitter, a beam deflector to control the beam, a microscope optic, a detection aperture, and detectors to detect the detection light or fluorescent light. The illumination light is coupled via a beam splitter. The fluorescent light or reflection light coming from the object returns to the beam splitter via the beam deflector, traverses this, and is subsequently focused on the detection aperture, behind which are located the detectors. Detection light that does not originate directly from the focal plane takes another light path and does not pass through the detection apertures so that one obtains point information by which a three-dimensional image is constructed by sequential scanning of the object. In most cases, a three-dimensional image is achieved fromdatafrom layered imaging.
The resolution of a confocal scanning microscope is, among other things, dependent on the intensity distribution and the spatial spread of the focus of the excitation light beam. One possibility for increasing the resolution is provided by STED (Stimulated Emission Depletion) microscopy as it was developed by Prof. Stefan Hell. Here, the lateral edge regions of the focus volume of the excitation light beam are illuminated with a light beam of another wavelength, the so-called stimulation light beam or by the de-excitation light beam, which is preferably emitted by a second laser, in order, stimulated there by the sample regions excited by the first laser (i.e., the fluorescent molecules located there), to be brought back to the ground state. Only the spontaneously emitted light from the regions not illuminated by the second laser are detected so that an overall improvement in resolution is achieved.
It has been demonstrated that it is possible to achieve an improvement in resolution both laterally and axially if the focus of the stimulation light beam can ideally be formed on the inside as a zero position, namely in all three spatial directions. Visually, this means that a hollow sphere is formed in the inside of the intensity profile of the stimulation light beam in which the intensity of the stimulation light beam is zero, i.e., no beam is present. The form of the stimulation focus results from the Fourier transforms of the phase filter function. A number of different phase filters are known. For example, there is the so-called vortex phase filter or axial phase filter as, for example, described in Appl. Phys. Lett., Vol. 82, No. 18, 5 May 2003, 3125-3127. Using an axial phase filter, the light that traverses the central part of the plate is delayed by half a wavelength opposite the edge region of the plate. In the process, the portion of the light that traverses the central region must be the same as the portion that traverses the edge region. This leads by means of interference effects to an extinguishing of the light beam in the central region. In practice, it is often not possible to achieve a very great improvement in resolution in both the lateral and in the axial direction equally because the ideal hollow sphere depicted is difficult to produce. Therefore, the decision is often made for an arrangement by which high resolution may be achieved either in the lateral or in the axial direction, depending on the particular application.
In addition, different wavelengths for the excitation and de-excitation light must be used for different fluorescent dyes. However, the known phase filters have a disadvantage in that they precisely implement only one phase shift which leads to the desired intensity distribution in the focus for only one particular light wavelength. They are therefore generally applicable for only a narrow wavelength band of approximately 60 nm around the central wavelength. This is, however, a great disadvantage because fluorescent dyes occur in a broad range of wavelengths, and various fluorescent dyes are used for certain biological applications. Then, however, because the phase filters are not attuned to this fluorescent dye, only poorer resolution is achievable.